Rho-kinase induces association of adducin with the cytoskeleton in platelet activation

We examined whether adducin function is regulated through Rho-kinase after agonist stimulation in platelets. A variety of stimuli such as thrombin, STA₂ (a stable analog of TXA₂), Ca²⁺ ionophore, phorbol diester, and shear stress induced phosphorylation of α-adducin at Thr445. Preincubation with the Rho-kinase inhibitor Y-27632 in platelets inhibited agonist-induced phosphorylation of α-adducin. STA₂ stimulation led to a redistribution of adducin from Triton-insoluble (high speed) fraction (membrane skeleton) to Triton-insoluble (low speed) fraction (cytoskeleton) and detergent-soluble fraction. Phosphoadducin at Thr445 was selectively isolated in the cytoskeletal fraction, whereas phosphoadducin at Ser726 was mainly present in the Triton-soluble fraction. Y-27632 inhibition of STA₂-induced α-adducin phosphorylation at Thr445 inhibited incorporation of α-adducin and spectrin into the platelet cytoskeleton, although Y-27632 did not affect phosphorylation of α-adducin at Ser726. These results suggest that Rho-kinase regulates the association of α-adducin and spectrin with the actin cytoskeleton in platelet activation.